Complex matrix for biomedical use

ABSTRACT

A complex matrix is constituted by at least one biocompatible polymer of natural origin, cross linked and to which are grafted small chains of a molecular weight less than 50,000 Da with an amount of grafting of 10 to 40%, as well as a process for preparation of a biocompatible matrix that is partly degradable, constituted by at least one polymer of natural origin, consisting: on the one hand, grafting small chains of molecular weight less than 10,000 Da, with a grafting amount of 10 to 40%, on the other hand, cross linking the principal polymer chains to create a homogeneous matrix.

The present invention relates to a biocompatible matrix, constituted byat least one polymer of natural origin, strongly functionalized,permitting the replacement of biological fluids, the separation oftissues or tissue increase. The matrix of the present invention ischaracterized by a long persistence in vivo, obtained by retarding itschemical, biological and mechanical degradation.

The present invention provides a process and compositions in the form ofa complex matrix of at least one polymer of natural. origin, to obtainmedical (pharmacologically active) devices adapted to increase thetissue separation or viscosupplementation, totally biodegradable butcharacterized by a long persistence in vivo.

The injection of a viscoelastic solution is often envisaged to replacethe natural synovial liquid which, in arthrosic patients, can no longerensure chondroprotective functions, lubrication and absorption of shocksgiven a reduction of the quantity of the molecular weight of theconstituent glycosaminoglycanes. These products are rapidly eliminatedfrom the synovial pocket.

The tissue increase is desired both in the case of therapeuticapplications and for cosmetic purposes.

In the case of therapeutic applications, certain tissues require beingenlarged to ensure their function; this can be the case of vocal cords,the esophagus, the urethral sphincter, other muscles . . . .

The patients can have recourse to aesthetic surgery for overcomingwrinkles, masking scars, increasing the lips . . . . But, in addition tothe high cost associated with this practice, the drawbacks are numerous,because it is an invasive and risky procedure. Injection of materialsadapted to increase tissue is a widely used method. The hypodermicneedles used as medical device have the advantage of being easy to use,precise, and constituting a non-invasive method.

The injectable materials available on the market are products eitherpermanent or biodegradable.

Non-Resorbable Permanent Products

There exist two approaches for using non-resorbable products: theinjection of silicone or a suspension of solid particles in a vectorsolution.

The injection of silicone has been widely used. However, given theundesirable long-term effects (nodules, ulcers of the skin), this methodis more and more abandoned [Edgerton et al. “Indications for andpitfalls of soft tissue augmentation with liquid silicone”. Plast.Reconstr. Surg, 58:157-163 (1976)].

The injection of solid microparticles also permits an increase ofpermanent tissue.

U.S. Pat. No. 5,344,452 discloses the use of a pulverulent solid,constituted by small particles, of a diameter comprised between 10 μmand 200 μm, and having a very smooth surface. Artecoll® and Arteplast®,products of commerce, are constituted by a suspension of microspheres ofpolymethacrylate in a collagen solution.

EP-A-1 091 775 proposes a solution of fragments of methacrylate hydrogelin a solution of hyaluronate. The particles of silicone, ceramics,carbon or metals (U.S. Pat. No. 5,451,406, U.S. Pat. No. 5,792,478, U.S.2002-151466), the fragments of polytetrafluoroethylene, of glass orsynthetic polymers (U.S. 2002-025340), and balls of collagen have alsobeen used but the results have been disappointing, given the secondaryreactions, and the biological degradation and the migration of theresidual products. Thus, the particles have least one of thesedrawbacks: a too great diameter or an irregular shape, which makes theparticles cling to each other, which can render the injection difficultthrough a fine needle, the too fragile particles can break duringinjection, the injection of too small particles leads to rapid digestionby the macrophages and other constituents of the lymphatic system, theinjected particles can move and not adhere to the environmental cells.

The permanent character of these products accordingly leads to majordrawbacks: the risk of activation of macrophages, the migration of thesynthetic fragments constituting the product or the appearance ofgranuloma which can require the injection of steroids, or even anexcision. Moreover, this type of product does not permit retouching ifnecessary.

Among the degradable biological materials, can be mentioned solutions ofcollagen or of cross-linked hyaluronic acid.

Collagen Corporation has developed a preparation based on collagencross-linked with glutaraldehyde (U.S. Pat. No. 4,582,640). This productis digested by the enzymatic or biochemical route, by macrophages,eliminated by the lymphatic system, and hence rapidly degraded. Repeatedtreatments are accordingly necessary.

U.S. Pat. No. 5,137,875 claims the use of aqueous suspensions orsolutions of collagen containing hyaluronic acid, but this productcannot constitute a solution for long term treatment.

EP 0 466 300 proposes the injection of a viscoelastic gel comprised by amatrix dispersed in a liquid phase, the two phases being composed byhylan, hyaluronate of high molecular weight of animal origin, crosslinked and extracted.

The hyaluronic acid esters and the cross linked derivatives ofhyaluronic acid have been developed for the purpose of increasing thetime of absorption of this glycosaminoglycane and hence obtaininggreater residence times. Among such products adapted for cosmetic use,can be cited Restylane®, a biphasic gel constituted by a fluid phase(non-cross-linked hyaluronate), and a very cross linked phase. If theintermolecular or intramolecular linkages of polysaccharides or estersof acid polysaccharides are used for numerous applications, for examplethe prevention of post-surgical adherence (EP 0 850 074, U.S. Pat. No.4,851,521, EP 0 341 745), these products cannot constitute a longpersisting effect given the high level of enzymatic degradation and thelow lifetime of the ester linkages which, contrary to ether linkages,are degradable in physiological environments (U.S. Pat. No. 4,963,666).

So as to increase the persistence of the matrix, it can be noted thatthe tendency is to use polymers of high molecular weight or to increasethe degree of cross linkage. But if the cross linkage increases in asubstantial manner the lifetime of the product, the manipulation ofthese highly cross-linked gels, and hence very constrained, is verydelicate because the other sites of the polymer not protected by a crosslinkage are mechanically and chemically rendered fragile and moresusceptible to being attacked.

Moreover, a large increase of the degree of cross linkage can lead toproducts that are difficult to inject.

EP 0 749 982 proposes grafting an antioxidant to a matrix with a lowrate of grafting.

It thus appears clearly that the existing materials do not provide anideal solution, and the search for new products for the increase oftissue, the separation of the tissues or the viscosupplementation,continues, with the aim of identifying highly biocompatible materials,easily used in the field of clinical use, having a lifetime such thatthis product disappears when its function is no longer needed, butsufficient to limit the medical and surgical interventions.

SUMMARY OF THE INVENTION

Although the conditions for increase, tissue separation andviscosupplementation have been known for many years, and numeroussolutions have been proposed for therapeutical and cosmeticapplications, the present invention provides a process and proposes newcompositions permitting the medical device to be effective long termwithout secondary effects. These same compositions can also prove to beuseful to constitute vectors for active pharmacological substances.

The principle of the present invention is based on the occupation of alarge number of sites of the polymeric chains to retard chemical andenzymatic attacks directly on the principal chain of the polymer. Thegrafting of small molecules coupled with cross linkage leads to increaseof the density of the matrix, and hence the time necessary for it todegrade, whilst limiting its agility induced by a too great degree ofcross linkage. The coupling of two types of functionalization,reticulation and grafting, also permits increasing the ease of use of amatrix adapted to be injected by recourse to a matrix which has the samenumber of sites occupied on the principal chain of the polymer but whosedegree of cross linkage is greater. The effect permitting the longpersistence of the composition can be amplified if the grafted moleculeshave antioxidant properties. Antioxidant agents can also be dispersed inthe matrix. The use of cellulosic derivatives or other polymersnaturally absent in the human being to constitute the product, alsopermits retarding the degradation of the matrix given the lack ofspecific hydrolases.

In the context of the present invention, the word “site” designated allthe points on the polymer chain adapted to be attacked; it can be amatter of pendant functional groups such as hydroxy or carboxy groups ora chain such as ether linkages.

The effect of long persistence of the medical device permits spacing themedical interventions and hence improving the quality of the life of thepatients.

Another object of the present invention is to provide a same compositioncontaining one or several therapeutically active molecules.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a biocompatible complex monophasic matrixwith long persistence, comprised by at least one highly functionalizedpolymer of natural origin. By long persistence, is meant an in vivolifetime greater than that of a product having an identical degree offunctionalization but obtained by another process than that of thepresent invention, characterized most often by a single cross linkage.

The substance adapted for viscosupplementation or tissue augmentation iscomprised by at least one polymer of a molecular weight greater than100,000 Da, selected from polysaccharides such as hyaluronic acid,chondroitine sulfate, keratane, keratane sulfate, heparin, heparinsulfate, cellulose and its derivatives, xanthanes and alginates,proteins, or nucleic acids, this polymer being highly functionalize bygrafting of small chains and a cross linkage permitting the creation ofa matrix. By matrix is meant a three-dimensional network constituted bypolymers of biological origin doubly functionalized by cross linking andgrafting.

The cross linking agent can be selected particularly from di- orpolyfunctional epoxides, for example 1,4-butanediol diglycidyl ether(also called 1,4-bis (2,3-epoxypropoxy)butane),1-(2,3-epoxypropyl)2,3-epoxy cyclohexane and 1,2-ethanediol diglycidylether, the epihalohydrins and divinylsulfone.

The degree of reticulation, defined as the ratio between the number ofmoles of reticulant ensuring the linkage of the chains of the polymerand the number of moles of structures of the polymer, is comprisedbetween 0.5 and 25% in the case of injectable products, from 25 to 50%in the case of solids.

So as to increase the steric size and the density of the matrix, andhence the time necessary for the product to be degraded by chemical andbiochemical action, small chains can be grafted by ionic linkages or ina covalent fashion, preferably by etherification, onto the matrix. Thesegrafted chains will occupy a large number of sites on the matrix, whichpermits increasing substantially the lifetime of the product withoutmodifying the mechanical or rheological character of the polymerconstituting the matrix. To the mechanical protection is added abiological and chemical protection constituted by “lures”.

The chains grafted on the functional groups of the hydroxy or carboxytype probably protect on the one hand directly these functional groupshaving reacted, and on the other hand indirectly the other sitesdetectable by steric hindrance.

The grafted chains and the polymers of natural origin of small sizecomprise more available attackable sites than the sites masked by thematrix, or polymers not recognized by the enzymes of the organism. Inthis latter case, it can be a matter of cellulosic derivatives or ofderivatives of other biopolymers not naturally present in the human bodywhich will not be degraded by the enzymes of the organism, but will besensitive to attack by the free radicals and other reactive radicals. Itcan for example be a matter of carboxymethylcellulose.

The grafted chains can moreover be unpolymerized chains havingantioxidant properties or properties to inhibit the reactions ofdegradation of the polymer matrix. It can for example be a matter ofvitamins, enzymes or cyclic molecules.

The amount of grafting which is defined as the ratio between the numberof moles of grafted molecules or the number of moles of the graftedpolymer and the number of moles of the structure of the cross linkedpolymer or polymers, is comprised between 10 and 40%.

The grafting of the chains of small size, which is to say of a size lessthan 50,000 Da, and preferably of the order of 10,000 Da or less, onnumerous sites of the polymer matrix, permits preserving the injectablecharacter of the final product because the amount of reticulation is notincreased, whilst the presence of these grafted chains prevents theattack of the matrix by the environmental medium and ensures a longerpersistence of the product after injection.

The grafted molecules can be grafted by covalent linkage to theprincipal chains, directly for example by esterification oretherification of the hydroxy or carboxy groups of by means of a bi- orpolyfunctional molecule selected from epoxids, epihalohydrins ordivinylsulfone.

Those skilled in the art will easily understand that such a process offunctionalization has significant advantages relative to a simple crosslinkage.

The grafting and cross linkage can take place at the same time, or thegrafting can precede the cross linkage, or vice versa.

So as to retard degradation by free radicals, a molecule havingantioxidant properties may also be dispersed in the stronglyfunctionalized matrix.

For example, vitamin C, a slender hydrosoluble molecule havingantioxidant properties, can be used in the case of non-inflamed tissuesto avoid the oxidation of the organic macromolecules, to capture thefree radicals, but also to stimulate the synthesis of the extracellularmatrix, particularly of collagen. This effect can be particularlyinteresting in the case of dermatological and cosmetic applications, toimprove the elasticity of the skin.

Vitamin A, which has numerous advantages (antioxidant action, influenceon the development of tissues and participation in the treatment of theskin) could also be dispersed in this highly modified matrix which, byits density, would permit progressive release of the activepharmacological agent.

Melatonin, which would be released in a very small quantity, is apowerful antioxidant agent and regenerator of the skin and defender ofthe immune system which could also be dispersed in the matrix.

So as to retard enzymatic degradation, the use of polymers not naturallyavailable in the human body such as cellulosic derivatives, particularlycarboxymethylcellulose, is recommended in the composition of matrices ofthe present invention, given the absence of specific hydrolases of thesepolymers.

As a result, the long persistence effect of the products of the presentinvention is obtained by greatly increasing the steric hindrance, byblocking a very large number of “attackable” sites biologically andchemically without rendering the other sites fragile, thanks to the useof the grafting of short chains and a quantity of cross linkage whichremains fairly low compared to other products presently on the market.

Moreover, this type of functionalization permits for a number ofidentical occupied sites on the principal chains of the constituentpolymer of the matrix, an injectability facilitated relative to that ofgels modified by cross linkage alone.

FIG. 1 shows the much slower degradation as a function of time, ofinjectable products according to the present invention, and two productsavailable on the market, Juvéderm® and Restylane® (composition ofpolysaccharide gel of U.S. Pat. No. 5,827,937).

The invention also relates to a complex matrix constituted by at leastone biocompatible polymer of natural origin, cross linked and to whichare grafted chains of molecular weight less than 50,000 Da with aquantity of grafting of 10 to 40%.

The biocompatible polymer of natural origin constituting the matrix ispreferably selected from polysaccharides such as hyaluronic acid,chondroitine sulfate, keratane, keratane sulfate, heparin, heparanesulfate, cellulose and its derivatives, xanthanes and alginates,proteins, or nucleic acids.

According to a preferred embodiment, the biocompatible polymer ofnatural origin is a polymer not naturally present in the human body,such as a cellulosic derivative, a xanthane or an alginate, which iscross linked with at least one polymer naturally present in the humanbody selected from polysaccharides such as hyaluronic acid, chondroitinesulfate, keratane, keratane sulfate, heparin, heparin sulfate, xanthanesand alginates, proteins or nucleic acids.

Preferably, the amount of cross linkage, defined as the ratio betweenthe number of moles of reticulating agent ensuring the linkage of thepolymer chains and the number of molds of polymer structure, iscomprised between 0.5 and 50%, in particular between 0.5 and 25% in thecase of injectable products, and between 25 and 50% in the case of solidproducts. The cross linking agent ensuring the linkage of the chain canbe provided by a bi- or polyfunctional molecule selected from epoxydes,epihalohydrines and divinylsulfone.

The matrix can contain antioxidant agents, vitamins or otherpharmaceutically active dispersed agents.

The invention also relates to the use of the matrix defined above toreplace, fill or supplement a biological fluid or tissues.

The invention also relates to a process to obtain a biocompatible matrixwhich is partly biodegradable, constituted by at least one polymer ofnatural origin, characterized in that it consists:

-   -   on the one hand in grafting small chains of molecular weight        less than 50,000 Da with a grafting quantity of 10 to 40%,    -   on the other hand, cross linking the principal chains of the        polymer to create a homogeneous matrix.

EXAMPLES

Examples are provided to illustrate the invention, but in no case dothey limit the scope of the invention.

First Series of Examples (Examples 1 to 3) Example 1—(Cross Linkage)

150 mg of sodium hyaluronate (M.W.=2×10⁶ Da) and 50 mg ofcarboxymethylcellulose (M.W.=2×10⁵ Da) are added to 6 ml of 0.5% soda.The whole is homogenized in a mixture until a transparent solution isobtained. 10 μl of 1,4-butanediol diglycidyl ether (BDDE) are then addedto the solution and the whole is mixed for 12 hours at 20° C. The pH isadjusted to physiological pH. The obtained matrix is then dialyzed for24 hours (regenerated cellulose, limit of separation,M.W.=12,000-14,000) against a solution of phosphate buffer at pH 7 (gel1).

Example 2—(Cross Linkage)

150 mg of sodium hyaluronate (M.W.=2×10⁶ Da) and 50 mg ofcarboxymethylcellulose (M.W.=2×10⁵ Da) are added to 6 ml of 0.5% soda.The whole is homogenized in a mixture to obtain a transparent solution.20 μl of 1,4-butanediol diglycidyl ether (BDDE) is then added to thesolution and the whole is mixed for 12 hours at 20° C. The pH isreadjusted to physiological pH. The obtained matrix is then dialyzed for24 hours (regenerated cellulose, limit of separation,M.W.=12,000-14,000) against a phosphate buffer solution at pH 7 (gel 2).

Example 3—(Cross Linkage and Grafting)

150 mg of sodium hyaluronate (M.W.=2×10⁶ Da) and 50 mg ofcarboxymethylcellulose (M.W.=2×10⁵ Da) are added to 6 ml of 0.5% soda.The whole is homogenized in a mixture until a transparent solution isobtained. 20 μl of 1,4-butanediol diglycidyl ether (BDDE) is then addedto the solution and the whole is mixed for 8 hours at 20° C. 40 mg ofbenzyl hyaluronate (esterified to 75%, M.W.=10⁴ Da) are added and mixedfor 2 hours at 20° C. 10 mg of vitamin C is then added and incorporatedin the viscous matrix. The pH is adjusted to physiological pH. The wholeis then mixed for 2 hours. The obtained matrix is then dialyzed for 24hours (regenerated cellulose, limit of separation, M.W.=12,000-14,000)against a solution of phosphate buffer at pH 7 (gel 3).

Calculation of the Amount of Grafting: $\begin{matrix}{{{Quantity}\quad{of}\quad{grafting}} = \frac{\left( \left( {{m_{vitC}/M_{vitC}} + \left( {m_{HAbenzyl}/M_{HAbenzyl}} \right)} \right) \right.}{\left( {\left( {m_{HA}/M_{HA}} \right) + \left( {m_{CMC}/M_{CMC}} \right)} \right)}} \\{0.246\left( {{which}\quad{is}\quad{to}\quad{say}\quad 24.6\quad\%} \right)}\end{matrix}$wherein:

-   -   m: weight in g    -   M: molecular weight of the polymer unit in g/mol    -   Vit C: vitamin C    -   HA: hyaluronate    -   HAbenzyl: benzyl hyaluronate    -   CMC: carboxymethylcellulose

The amount of grafting, calculated by supposing that the carboxylicfunctions are all in the form of sodium salt and that thecarboxymethylcellulose has a quantity of substitution of 0.9, is 24.6%.

Rheological studies have shown a slower decrease of these properties forthe gel of example 2 (gel 2) than for that of example 1 (gel 1) whenthese gels are held at 37° C. Although an in vivo study has not beencarried out to date, the degradation of gel 2 is probably slower thanthat of gel 1, which itself must be degraded less rapidly than asynthesized gel according to the same process but comprised exclusivelyof sodium hyaluronate. This result is suggested by the data concerningthe in vivo lifetime of the unreticulated carboxymethylcellulose,compared to that of unreticulated sodium hyaluronate injected in thesame concentration and having a comparable molelcular weight.

Gel 2 has a lifetime greater than that from the first example thanks toa degree of cross linkage twice as high.

The number of sites occupied in the gel of example 3 (gel 3) is at leastequal to that of gel 2 and the decrease in the viscosity of gel 3 in thecourse of time is slower than that of gel 2 (when these gels are held at37° C.).

Second Series of Examples (Examples 4 to 7) Example 4—(Cross Linkage)

1 g of sodium hyaluronate (M.W.=2×10⁶ Da) is placed in 10 ml of a sodasolution at 1%. The whole is homogenized with a mixture until thesolution becomes transparent. 100 μl of 1,4-butanediol diglycidyl ether(BDDE) is then added and the whole is again mixed for 2 hours at 50° C.The solution is adjusted to physiological pH and the volume isreadjusted to 50 ml with a phosphate buffer. The obtained matrix is thendialyzed for 24 hours (regenerated cellulose, limit of separation,M.W.=12,000-14,000) against a phosphate buffer solution at pH 7 (gel 4).

Example 5—(Cross Linkage)

1 g of sodium hyaluronate (M.W.=2×10⁶ Da) is placed in 10 ml of a 1%soda solution. The whole is homogenized with a mixture until thesolution becomes transparent. 130 μl of 1,4-butanediol diglycidyl ether(BDDE) is then added and the whole is again mixed for 2 hours at 50° C.The solution is adjusted to physiological pH and the volume isreadjusted to 50 ml with a phosphate buffer. The obtained matrix is thendialyzed for 24 hours (regenerated cellulose, limit of separation,M.W.=12,000-14,000) against a phosphate buffer solution of pH 7 (gel 5).

Example 6—(Cross Linkage)

0.8 g of sodium hyaluronate (M.W.=2×10⁶ Da) and 0.2 g ofcarboxymethylcellulose (M.W.=3×10⁵ Da) are placed in 10 ml of a 1% sodasolution. The whole is homogenized with a mixer until the solutionbecomes transparent. 130 μl of 1,4-butanediol diglycidyl ether (BDDE) isthen added and the whole is again mixed for 2 hours at 50° C. Thesolution is adjusted to physiological pH and the volume is readjusted to50 ml with a phosphate buffer. The obtained matrix is then dialyzed for24 hours (regenerated cellulose, limit of separation,M.W.=12,000-14,000) against a phosphate buffer solution of pH 7 (gel 6).

Example 7—(Cross Linkage and Grafting)

0.8 g of sodium hyaluronate (M.W.=2×10⁶ Da) and 0.2 g ofcarboxymethylcellulose (M.W.=3×10⁵ Da) are placed in 10 ml of a 1% sodasolution. The whole is homogenized with a mixer until the solutionbecomes transparent. 130 μl of 1,4-butanediol diglycidyl ether (BDDE)are then added and the whole is mixed for 1 hour 20 minutes at 50° C.0.2 g of heparin (M.W.=3×10³ Da) diluted in 4 ml of 0.5% soda solutionis then added to the gel in the course of formation and the whole isagain mixed. The mixture is brought to physiological pH and the volumeis readjusted to 50 ml with a phosphate buffer. The obtained matrix isthen dialyzed for 24 hours (regenerated cellulose, limit of separation,M.W.=12,000-14,000) against a phosphate buffer of pH 7 (gel 7).

Computation of Amount of Grafting:Amount of grafting=(m _(heparin) /M _(heparin))/((m _(HA) /M _(HA))+(m_(CMC) /M _(CMC)))=10.3%wherein:

-   -   m: weight in g    -   M: molecular weight of the polymer unit in g/mol    -   HA: hyaluronate    -   CMC: carboxymethylcellulose

The quantity of grafting, calculated by supposing that half theionizable functions are in the form of sodium salt and that thecarboxymethylcellulose has a substitution amount of 0.9, is 10.3%.

Moreover, a process has been set forth to quantify the injectability ofthe different gels obtained in examples 1 to 7. This process uses themeasurement of the force necessary for the ejection of the differentgels obtained through a needle of type 27 G. Each obtained gel is placedin a syringe of 1 ml whose outlet is provided with a needle of type 27G. The syringe is held vertical by a carrier and a weight is thenapplied to the piston of the syringe, at a constant speed defined by theuser. A detector measures the force necessary to eject the product. Inthe first series of examples, the speed of ejection is 75 mm/min and inthe second series of examples, the speed of ejection is 15 mm/min.

The values of the force of ejection measured for the gels of examples 1to 7 is given in tables 1 and 2 hereafter. TABLE 1 Force of ejectionGels V = 75 mm/min 1 (cross linkage) 20N +/− 4N 2 (cross linkage) 32N+/− 4N 3 (cross linkage and 25N +/− 4N grafting)

According to the results given in the table, for an equivalent amount ofcross linkage, the cross linked and grafted gels according to theinvention have a force of ejection less (and hence a betterinjectability) than that of the cross linked gels (comparison of gels ofexample 2 and example 3). TABLE 2 Force of ejection Gels V = 15 mm/min 4(cross linkage) 14N +/− 4N 5 (cross linkage) 23N +/− 4N 6 (crosslinkage) 26N +/− 4N 7 (cross linkage and 24N +/− 4N grafting)

As previously observed, an increase in the amount of cross linkage leadsto increase of the force necessary to eject the product (comparison ofgels 4 to 6). At an identical amount of cross linkage, thisinjectability is more difficult for cross linked gels HA/CMC. But if theinjectability is higher, the persistence of these gels must also belonger. The last example (comparison of gels 6 and 7) emphasizes thefact that the grafting of small chains of heparin permits decreasing theforce necessary for ejection whilst protecting the cross linked matrix,by steric hindrance and by the biological properties of this polymer.

1-11. (canceled)
 12. Complex matrix constituted by at least onebiocompatible polymer of natural origin, cross linked with a crosslinking agent of a bi- or polyfunctional molecule selected fromepoxydes, epihalohydrines and divinylsulfone and on which are graftedchains of molecular weight less than 50,000 Da, selected from polymersof natural origin of small size, preferably cellulosic derivatives orother derivatives of biopolymers not naturally present in the human bodyand/or non-polymeric chains having antioxidant properties or propertiesfor inhibiting reactions of degradation of said matrix, preferablyvitamins, enzymes or molecules comprising one or several cycles, with aquantity of grafting, defined as being the ratio between the number ofmoles of grafted molecules and the number of moles of units of thepolymer, from 10 to 40%.
 13. Matrix according to claim 12, in which thebiocompatible polymer of natural origin is selected from hyaluronicacid, chondroitine sulfate, keratane, keratane sulfate, heparin, heparinsulfate, cellulose and its derivatives, xanthanes and alginates,proteins, or nucleic acid.
 14. Matrix according to claim 12, in whichthe biocompatible polymer of natural origin is a polymer not naturallypresent in the human body such as a cellulosic derivative, a xanthane oran alginate which is cross linked with at least one polymer naturallypresent in the human body selected from hyaluronic acid, chondroitinesulfate, keratane, keratane sulfate, heparin, heparane sulfate,xanthanes and alginates, proteins or nucleic acids.
 15. Matrix accordingto claim 12, in which the amount of cross linkage, defined as the ratiobetween the number of moles of the cross linking agent ensuring thelinking of the polymer chains and the number of moles of units of thepolymer, is comprised between 0.5 and 50%, in particular between 0.5 and25% in the case of injectable products, and between 25 and 50% in thecase of solid products.
 16. Matrix according to claim 12, containingantioxidant agents, vitamins and other dispersed pharmacologicallyactive agents.
 17. Matrix according to claim 12, containing vitamins orother dispersed pharmacologically active agents.
 18. A method toseparate, replace, fill or supplement a biological fluid or tissuescomprising a step of applying an effective amount of a matrix accordingto claim
 12. 19. Process for the preparation of a partly biodegradablebiocompatible matrix constituted by at least one polymer of naturalorigin, characterized in that it consists in: on the one hand graftingsmall chains of molecular weight lower than 50,000 Da with an amount ofgrafting of 10 to 40%, the small chains being selected from polymers ofnatural origin of small size, preferably cellulosic derivatives orderivatives of other biopolymers not naturally present in the human bodyand/or unpolymerized chains having antioxidant properties or propertiesof inhibiting reactions of degradation of said matrix, preferablyvitamins, enzymes and molecules comprising one or several units, on theother hand cross linking the principal chains of the polymer to create ahomogeneous matrix, with the help of a cross linking agent which is abi- or polyfunctional molecule selected from epoxydes, epihalohydrinesor divinylsulfone.
 20. Matrix according to claim 13, in which thebiocompatible polymer of natural origin is a polymer not naturallypresent in the human body such as a cellulosic derivative, a xanthane oran alginate which is cross linked with at least one polymer naturallypresent in the human body selected from hyaluronic acid, chondroitinesulfate, keratane, keratane sulfate, heparin, heparane sulfate,xanthanes and alginates, proteins or nucleic acids.
 21. Matrix accordingto claim 13, in which the amount of cross linkage, defined as the ratiobetween the number of moles of the cross linking agent ensuring thelinking of the polymer chains and the number of moles of units of thepolymer, is comprised between 0.5 and 50%, in particular between 0.5 and25% in the case of injectable products, and between 25 and 50% in thecase of solid products.
 22. Matrix according to claim 14, in which theamount of cross linkage, defined as the ratio between the number ofmoles of the cross linking agent ensuring the linking of the polymerchains and the number of moles of units of the polymer, is comprisedbetween 0.5 and 50%, in particular between 0.5 and 25% in the case ofinjectable products, and between 25 and 50% in the case of solidproducts.
 23. Matrix according to claim 13, containing antioxidantagents, vitamins and other dispersed pharmacologically active agents.24. Matrix according to claim 14, containing antioxidant agents,vitamins and other dispersed pharmacologically active agents.
 25. Matrixaccording to claim 15, containing antioxidant agents, vitamins and otherdispersed pharmacologically active agents.
 26. Matrix according to claim13, containing vitamins or other dispersed pharmacologically activeagents.
 27. 16. Matrix according to claim 14, containing vitamins orother dispersed pharmacologically active agents.
 28. Matrix according toclaim 15, containing vitamins or other dispersed pharmacologicallyactive agents.
 29. Matrix according to claim 16, containing vitamins orother dispersed pharmacologically active agents.
 30. A method toseparate, replace, fill or supplement a biological fluid or tissuescomprising a step of applying an effective amount of a matrix accordingto claim
 13. 31. A method to separate, replace, fill or supplement abiological fluid or tissues comprising a step of applying an effectiveamount of a matrix according to claim 14.